Process for producing cyclodextrins

ABSTRACT

A novel process for producing cyclodextrin characterized by subjecting starch to the activity of cyclodextrin glycosyltransferase produced by cultivation of Bacillus sp. No. 38-2, Bacillus sp. No. 135, Bacillus sp. No. 169, Bacillus sp. No. 13 or Bacillus sp. No. 17-1, at a pH of between 6.0 and 10.5, thereby to hydrolyse the starch.

United States Patent Horikoshi et a1. 1 1 Dec. 2, 1975 [5 1 PROCESS FORPRODUCING 3,826,715 7/1974 Horikoshi et a1 195/66 R CYCLODEXTRINS [75]Inventors: Koki Horikoshi, Fujimi, Japan FOREIGN PATENTS 0R APPLICATIONS7.109.224 9/1971 Japan 195/31 R 1 1 Asslgfleel Rlkagaku Kenkyusho, Japan2.044.984 3/1971 Germany 195/65 [22] Filed: Mar. 18, 1974 1 PP Nod452,139 Primary Examiner-Lionel M. Shapiro Assistant E.raminer-T. G.Wiseman [30] Foreign Application Priority Data Mar. 19. 1973 Japan48-31680 [57] ABSTRACT Dec. 6, 1973 Japan 48-136664 A novel process forproducing cyclodextrin character- [52] Cl 195/31 R; 95/62; 195/66 R?ized by subjecting starch to the activity of cyclodextrin 426/215g1ycosy1-transferase produced by cultivation of Bacil- [51] Int. Cl.C12D 13/02 lus 33-2 Bacillus NO, 35 BaCmuS Sp No, [58] Fleld of Searchm195/31 R, 66 R, 65, 62 9 Bacillus Sp No 13 or i l SP No, 17 at a pH ofbetween 6.0 and 10.5- thereby to hydrolyse the [56] References Citedstarch UNITED STATES PATENTS 3,812,011 5/1974 Okada et a1 195/31 R 4Claims, 5 Drawing Figures US. Patent Dec. 2, 1975 Sheet 2 of2 3,923,598

FIG.4

disfou FIG.5

PROCESS FOR PRODUCING CYCLODEXTRINS BACKGROUND OF THE INVENTION:

It is well known that dextrin is produced from starch by hydrolysis withamylase and it has usually a chain structure.

Regarding cyclodextrin, only Schardinger dextrin which has been producedby Bacillus macerans has been known in the Enzyme Hand Book edited in1970 by Shiro Akabori and published from Asakura Publishing Co. Thoughthe Schardinger dextrin is produced by a cyclodextringlycosyltransferase, said cyclodextrin glycosyltransferase has variousdefects including an extremely narrow optimum pH range (merely from 6.8to 7.0), low thermal stability (the optimum temperature is 40C) and lowproductivity of the cyclodextrin, and therefore said amylase can not beutilized for commercial production of the cyclodextrin.

SUMMARY OF THE INVENTION The present invention relates to a novelprocess for producing cyclodextrin.

An object of the invention is to provide a novel process for producingvarious kinds of cyclodextrins having the different structures as shownin FIG. 1.

Another object of the invention is to provide an efficient process forproducing cyclodextrins, which can be carried out easily underconditions of broad pH range and wide temperature range.

Further object of the present invention is to provide an efficientprocess which can produce cyclodextrins with high yield.

The above and other objects can be accomplished by a process accordingto the present invention, which comprises subjecting starch to acyclodextrin glycosyltransferase obtained by cultivation of one of theBacillus named below, at a pH of betweem 6.0 and 10.5.

BRIEF EXPLANATION OF DRAWINGS FIG. 1 shows the structures of variouskinds of cyclodextrins which can be obtained according to the presentinvention;

FIG. 2 is a graph showing relative activities at different pH values ofthe amylase obtained from Bacillus sp. No. 38-2, in which the relativeactivity has been calculated on the assumption that activity at pH 9.0is 100;

FIG. 3 is a similar graph showing relative activities of amylasesobtained from Bacillus sp. No. I35 and No. 169;

FIG. 4 is a graph showing relative activities and relative yield ofcyclodextrin at different pH values of the amylase obtained fromBacillus sp. No. 13; and

FIG. is a graph showing relative activities and relative yield ofcyclodextrin at different pH values of the amylase obtained fromBacillus sp. No. 17-1.

DETAILED DESCRIPTION OF THE INVENTION As described above the inventionof the present application is characterized by employing a specific cyclodextrin glycosyl transferase, namely one obtained by cultivation of aspecific amylase-producing microorganism which can grow only in analkaline medium.

The microrganism which can produce the cyclodextrin glycosyl-trsnsferasedescribed above includes Bacillus species No. 382, Bacillus species No.135, Bacillus species No. 169, Bacillus species No. 13 and Bacil- 2 lusspecies No. l7-1, all of them having been found by the inventors of thepresent invention.

Bacillus sp. No.38-2, No. and No. l69 have been isolated from the soilcollected in Hirosawa district of Wako-shi, Saitama Prefecture, Japan. I

Bacillus sp. No. 13 and No. 17-1 have been isolated from the soilcollected in Karuizawa-shi, Nagano Prefecture, Japan.

Characteristics and properties of those microorganisms will be disclosedbelow. However we have no in tention to restrict the invention to thosemicroorganisms, because any natural or artificial mutant, variant andother species can be used as far as the strain can produce thecyclodextrin producing alkaline amylase.

We have examined the properties and characteristics of the abovedescribed species according to the methods described in AerobicSpore-forming Bacteria by Nathan R. Smith, R. E. Gordon and F. E. Clark(United States Department of Agriculture, November 1952) and BergeysManual of Determinative Bacteriology (1957). Results of examination areshown as follows:

1. Bacillus sp. No. 38-2 a. Growth on Various Media b. MicroscopicMorphology:

Size of the microorganism is 0.5 0.6g. X 2.0 3.041.; The spore which isformed near the end of the cell is oval and has a size of 0.9 1.0;].X 1. 2 l.5p.; The sporangium is definitely swollen; The bacteria havepertrichous flagella and are motile and form motile collonies; Grampositive and non acid-fast.

It grows very well on an alkaline medium comprising soluble starch,yeast extract, peptone, K HPO and MgSO -7H O and containing 1% Na COwhereas growth on any neutral medium is poor.

c. Physiological Properties:

1,. Optimum Growth Condition:

pH around 10 Temperature: 37 40C Aerobic 2. Conditions under which themicroorganism grows:

pH 7.5 ll

Temperature up to 45C 3. Voges-Proskauer reaction Positive I NitrateReduced Catalase reaction Positive Gelatine, Casein Liquefied Hydrolysisof Starch Positive Utilization of Citrate Utilized but poor 9.Utilization of Ammonium Salt: Utilized 10. Growth in 7% Sodium ChlorideSolution Poor 11. Growth on Glucose-nitrate Medium Growth 12. Growthunder Anaerobic Condition Growth 13. Growth on Glucose-asparagine MediumGrowth 14. Production of lndole Negative 7 d. Utilization of CarbonSource:

3 Glucose, fructose, xylose, sucrose, maltose, lactose and arabinose areutilized, but galactose, trehalose and inulin are not utilized.Production of acid is observed.

2. Bacillus sp. No. 135: a. Growth on Various Media:

b. Microscopic Morphology:

Size of the microorganism is 0.6 0.8;; X 2.5 X 4 1.; The sporangium isslightly swollen and the spore is oval having size of 1.0 1.2;. X 1.5l.8p.. The microorganism has pertrichous flagella and motile. ThisBacillus grows very well on an alkaline medium comprising solublestarch, yeast extract, peptone, K HPO MgSO -7- H and containing 1% of NaCO and appears white. The characteristic of the species is that it growsvery well on alkaline medium, though grows a little on neutral medium.

c. Physiological Properties:

1. Optimum Growth Condition:

pH around 10 Temperature 37 40C Aerobic 2. Conditions under which thebacteria can grow:

Temperature: up to 42C Aerobic Gram Stainability PositiveVoges-Proskauer reaction Positive Nitrate Reduced Catalase PositiveGelatine and Casein Liquefied Hydrolysis of Starch PositiveUtilizationof Citrate Not utilized 10. Utilization of Ammonium SaltUtilized 11. Growth in 7% NaCl Solution No detected 12. Growth onGlucose-Nirtate Medium Growth scant 13. Growth under Anaerobic ConditionDetected 14. Production of Gas in Nitrate Medium under AnaerobicCondition No produced 15. Growth on Glucose-Asparagine Medium Growth d.Utilization of Carbon Source:

Glucose, mannose, salicin, cellobiose, lactose, sucrose, arabinose,mannitol and xylose are utilized, but production of acid can not beobserved, because a lot of carbonate is used.

3. Bacillus sp. No. 169:-

a. Growth on Various Media:

Table 3 Growth at Medium pH 7 pH 10 l. Bouillon Growth scant Poor growth2. Bouillon-agar Growth scant Poor growth 3. Glucose-Bouillon Growthscant Turbid. good growth 4. Glucose-Bouillon- Growth scant Good growthagar Gelatine medium Peptone water Potato medium Good growth liquefiedGrowth Poor growth Good growth b. Microscopic Morphology:

Size of the microorganism is 0.5 0.6;]. X 2 3p.; The sporangium isslightly swollenand the spore is oval having a size of 1.0 1.2 2 X 1.31.711.. The microorganism has pertrichous flagella and is motile. Itgrows very well on an alkaline medium comprising soluble starch, yeastextract, peptone, K HPO, and MgSO -7H O and containing 1% Na CO but ithardly grows on any neutral medium.

c. Physiological Properties:

1. Optimum growth condition:

Temperature 37 Aerobic.

2. Conditions under which the bacteria can grow:

Temperature up to 45C Aerobic.

. Gram stainability Positive, Changeable Voges-Proskauer reactionPositive Nitrate Reduced Catalase reaction positive Hydrolysis ofgelatin and casein Positive Hydrolysis of starch Positive Utilization ofcitrate Not utilized 10. Growth in 7% NaCl solution Not growth 1 1.Growth on glucose-nitrate medium Scant 12. Growth under anaerobiccondition: Growth 13. Production of gas in nitrate medium underanaerobic condition Not produced 14. Growth on glucose-asparagine mediumNot growth 15. Production of indol Negative (1. Utilization of CarbonSource:

Glucose, mannose, cellobiose, lactose, sucrose, mannitol and salicin areutilized very well, but arabinose and xylose are not utilized.

Production of acid can not be determined since the medium contains a lotof carbonate.

4. Bacillus sp. No. 13:

a. Growth on Various Media:

Table 4 Growth at Medium pH 7.0 pH 10.0

2. Bouillon-agar Growth scant plane 3. Bouillon-agar Growth scant slant4. Bouillon-gelatin Growth scant;

thrust not liquefaction of gelatin 1% Nn CO is added to the medium inorder to adjust pH value to 10.

b. Microscopic Morphology: Size of the cell is 0.5 0.7 1. X 2.0 4.0 1.;oval spore is formed at the end of the cell; size of the spore is 1.3

1.4;1. X 1.5 1.6 1.; the sporangium is definitely swollen; themicroorganism has pertrichous flagella and is motile. The gramstainability thereof is positive and the acid-fast test is negative.

6 size of a spore is 0.8 1.0;1. X 1.2 1.5 1.. The sproangium isdefinitely swollen. The microorganism has pertrichous flagella and ismotile; gram positive and non acid-fast.

Note: Above morphological observation has been 5 The above observationhas been made on a medium made on a medium comprising grams sodiumcarcomprising 10 grams sodium carbonate, 5 grams pepbonate, 5 gramspeptone, 5 grams yeast extract, 20 tone, 5 grams yeast extract, 20 gramsstarch, 1 gram grams starch, 1 gram K HPO 0.2 gram MgSO -7- K HPO 0.2gram MgSO .7H- O, grams agar and 1 H 0, 15 grams agar and 1 liter water.liter of water.

c. Physiological Properties: 10 C. Physiological Properties:

The following results have been observed on a media The followingresults have been observed on media which has been described in AerobicSpore-forming described in Aerobic Spore-forming Bacteria by N.Bacteria" by N. R. Smith, et al., modified by adding R. Smith, et al.,and modified by adding 1% Na CO re- 1% Na CO respectively. spectively.

1. Nitrate Reduced 15 l. Nitrate Reduced 2. Denitrogenation Negative 2.Denitrogenation Negative 3. Methyl red test No change of color, due tobasic- 3. Methyl Red reaction No change of colour, clue to ity of themedium basicity of the medium.

4. Voges-Proskauer reaction Positive 4. Vogel-Proskauer reactionNegative 5. Production of indole Negative 5. Production of indoleNegative 6. Production of hydrogen sulfide: Negative 6. Production ofhydrogen sulfide Negative 7. Hydrolysis of starch Positive 7. Hydrolysisof starch Positive 8. Utilization of citric acid Utilized 8. Utilizationof citric acid Utilized very well 9. Utilization of nitrate and ammoniumSlightly uti- 9. Utilization of nitrate and ammonium salt Utilized lizedvery well 10. Production of pigment Negative 10. Production of pigmentNot produced 11. Catalase reaction Positive 11. Catalase reactionPositive 12. pH range for growth 7.5 11 12. pH range for growth 7 11 13.Temperature range for growth up to 42C; opti- 13. Temperature range forgrowth up to 42C, optimum 37 40C mum: 3740c 14. Behavior to oxygenAerobic 14. Behavior to oxygen Aerobic 15. Growth in 5% NaCl solutionSlightly growth 15. Growth in 7% NaCl solution Growth well d.Utilization of Carbon Source: 0. Utilization of Carbon Source:

Lactose, arabinose, xylose, glucose, mannose, inosi- Lactose, arabinose,xylose, glucose, mannose, inocitole, fructose, galactose, maltose,sucrose, trehalose, tole, fructose, galactose, maltose, sucrose,trehalose, mannittol, starch, sorbitol and glycerine are utilized,mannitol,strach, sorbitol and glycerine are utilized and and acids areproduced. acids are produced. Production of gas has not detected.

Production of gas is not detected. Studying the bacteriologicalproperties disclosed 5. Bacillus sp. No. 17-1: above, Bacillus speciesNo. 38-2, Bacillus species No.

a. Growth on various media: 40 135, Bacillus species No. 169, Bacillusspecies No. 13

T and Bacillus species No. 17-1 shall belong to the Bacilable 5 lusgenus, because those miroorgamsms are an aerobic Gmwth and spore-formingbacteria respectively. Medim PH PH Further we found that foridentification of these mi- 1. Bouillon liquor Scant Growth, turbid andsedicroorganisms, Bacillus polymixa, Bacillus macerans and 2Bouillomagar slightly g fi g g'g figg Bacillus circulans shall beselected as known speeies for plane growth Smooth, brilliant,comparison, because every sproangium thereof 15 defiilfiglranslucemimilky nitely swollen. Even our new microorganisms are simi- 3.Bouillon-agar Slightly Spreading; milky white, in some Properties to theknown Species, y are growth translucent. entirely different incharacteristic properties, particu- 4. aoumomgelafln scam fi z f'zgl 'ggzg larly by the fact that the optimum pH value of our mithrust Noliquecroorganisms reside in alkaline side, whereas that of ficatim knownspecies in neutral.

1% Na,CO, is added to the medium in order to regulate pH value to 10.0.The following Table 6 shows various characteristic properties ofBacillus polymixa, Bacillus macerans and b Microscopic p gy Bacilluscirculans (known species) aswell as Bacillus The vegitative cell is arod having a size of 0.5 0.7g. N Baclnus Baclnus X 2.0 4.0a. Oval Sporeis formed at subterminal. The 3223" 13 and Baclnus 9 (new Table 6 Growthin Utilization Utilization Growth under Reduction Bacillus species 5%NaCl of citric of nitrate anaerobic conof nitrate acid dition Bacilluspalymixa Bacillus maceranr Bacillus circulans +or Bacillus sp. 38-2 IBacillus sp. Na.l35 j:

Table 6-continued Growth in Utilization Utilization Growth underReduction Bacillus species NaCl of citric of nitrate anaerobic conofnitrate acid dition Bacillus sp. I69 i Bacillus sp. No.13 i Bacillus5;). N0. l7-I -ll- +1- -H- From the above table it is noted that notonly every species of Bacillus sp. No. 38-2, No. 135, No. 169, No. 13and No. 17-1 has different characteristics from the known three species,but also they are not identical each other. Judging differences shown inthe table, we concluded that every species of Bacillus sp. No. 38-2,Bacillus sp. No. 135, Bacillus sp. No. 169, Bacillus sp. No. 13 andBacillus sp. No. 17-1 shall be identified as a new species respectively.

The strains identified as said Bacillus sp. No. 38-2, Bacillus sp. No.135, Bacillus sp. No. 169, Bacillus sp. No. 13 and Bacillus sp. No. 17-1have been deposited with the American Type Culture Collection (ATCC) at12301 Parklawn Drive, Rockville, Md. 20852 U.S.A. as ATCC access numbers21783, 21595, 21594, 31006 and 31007, in unrestricted conditionpermitting the public to have full access to the cultures, as of Mar.27, 1972, Aug. 19,1970, Aug. 19, 1970, Feb. 21, 1974 and Feb. 21, 1974,respectively. All restrictions on the availability of the culturedeposit to the public will be irrevocably removed upon the granting of apatnet from this application. Further, the above cultures will bemaintained by the depositor throughout the effective life of the patent.

The medium to be used for cultivation of the microorganism abovedescribed must be a basic medium containing carbonate, though it may beany of solid or liquid medium. Thus a medium comprising essentialcomponents for growth of the microorganism, such as carbon source,nitrogen source, inorganic salt and the like and containing addedcarbonate are used. Starch, soluble starch and the like are used as thecarbon source. Yeast extract, peptone, corn-steep liquor and the likeare used as the nitrogen source. Thus a medium comprising solublestarch, peptone, yeast extract, K HPO MgSO -7H O and an added carbonateis used. Any carbonate selected from anhydrous sodium carbonate,potassium carbonate, sodium bicarbonate and the like may be used.

We found that it is very important to add a sufficient amount ofcarbonate to the medium to make the pH value of the medium alkaline.

The amount of the carbonate to be added to the medium shall bepreferably in the range of 0.5% to 1.5% by weight. This fact has beendetermined by the following experiments.

The experiments have been carried out using a standard neutral mediumcomprising 5 gs. peptone, 5 gs. yeast extract, 20 gs. starch, 1 g k HPO0.2 g MgSO -7- H 0, 15 gs. agar and 1 liter of water and modified mediacontaining various salts and carbonates in amount shown in the Table 7.

Media having pH value of have been prepared by adding sodium hydroxideto the neutral medium.

Each of the media is innoculated with the strain of Bacillus sp. No.17-1 and cultured with shaking at a temperature of 37C.

Growth of the microorganism has been observed by taking the culturebroth after 18 hours into a cuvette of 1 cm and measuring the absorbanceof light at 660 mu.

The yield of cyclodextrin has been determined by measuring the activityof the culture broth after 3 days cultivation under the conditiondescribed in later.

These results have been shown in the Table 7.

From the results shown in the table, it is noted that the presence of asuitable amount of the carbonate in the medium is indispensable toproduce the subject enzyme to be used in the present invention.

The cultivation of the microorganism described above can be carried outby means of conventional aerobic shaking culture or air bubblingculture. It is preferable to culture for 24 96 hours at a temperaturebetween 30C and 37C.

The resulting enzyme can be isolated from the culture broth by anyconventional method. For example, when the cultivation is finished, themicroorganism is removed from the culture broth and then afterneutralization of the broth with acetic acid or the like or without anyneutralization, the broth is treated with an organic solvent such asmethanol or ammonium sulphate to precipitate enzyme and then theprecipitated enzyme is separated from the liquid and dehydrated, therebyto obtain crude powdery enzyme. The resulted crude enzyme can be usedfor production of the cyclodextrin of the present invention as it is.

Purified enzyme can be obtained from the above crude enzyme as follows:The crude powdery enzyme is dissolved in water and the resultingsolution is dialyzed against water overnight. The solution is passedthrough a column of diethyl aminoethyl cellulose (DEAE Cellulose)equilibratedwith 0.01 M Tris HCl buffer solution of pH 9.0. Thus theenzyme in the solution is completely adsorbed in the cellulose. Theadsorbed enzyme is eluted by changing the concentration of NaCl in thebuffer solution from 0.01M to 0.5M.

The active fractions are collected and concentrated. Then theconcentrated solution is purified by gelfiltration chromatography usingSephadex G75 and Sephadex G-lOO (Sephadex" is a resistered trade name)and the resulted cake is freez-dried, thereby to obtain a purifiedenzyme powder.

The activity of the resulting enzyme is determined as follows.

0.05 ml of the enzyme solution with a suitable concentration is mixedwith 0.5 ml of 1% soluble starch solutionin 0.1M glycine buffer (pH10.5).

The resulting mixture is subjected to a temperature of 40C for 2 hours,after reaction has been completed the solution is neutralized withacetic acid and further heated at 100C for minutes.

The resulting solution is mixed with 50p.g of gluco amylase, thesolution is subjected to a temperature of 40C for one hour to decomposethe residual starch and the amount of glucose produced in the solutionis determined by means of the dinitrosalicylic acid method.

The same process is repeated except using water instead of the enzymesolution.

The difference of the determined amount of glucose shows the amount ofproduced cyclodextrin.

One unit of the enzyme was defined as that amount of enzyme producing 1mg of the cyclodextrin under the method described above.

The activity of the enzyme can be assayed by the iodine method asfollows:

The enzyme solution (0.01 ml), which has been suit-- ably diluted so asto reduce the absorbance at 700 mg by from 10% to is mixed with 0.2 mlof 0.2% potato starch aqueous solution and 0.2 ml of 0.1M acetic acidbuffer solution having pH value of 4.5, then the resulted mixture isheated at 40C for 10 minutes. After reaction, the resulted solution ismixed with 0.3 ml of 0.2M hydrochloric acid and then 3 ml of 0.005%iodine sulution. The absorbance at 700 my. of the sample is measured.

The physicochemical properties of the enzymes produced from Bacillus sp.No. 38-2 (ATCC 21783), Bacillus sp. No. 135 (ATCC 21595), Bacillus sp.No. 169 (ATCC 21594), Bacillus sp. No. 13 (ATCC 31006) and Bacillus sp.No. 17-1 (ATCC 31007) will be explained in detail.

1. Enzyme produced from Bacillus sp. No. 38-2 (ATCC 21783) 1. SubstrateSpecificity:

the enzyme produced by cultivation of the above identified microorganismunder the specific conditions described above is active to starch, andreduces the starch-iodine reaction. However, reducing power can not bedetected. Then the enzyme is determined to be a liquefying amylase.

2. Optimum pH:

The optimum pH of the enzyme has been determined by measuring activityof the enzyme at various pH values by means of the method describedabove.

Each pH value has been achieved by use of the following buffersrespectively.

pH Buffer solution 4 5 Acetate 5 8.5 Tris-maleate 9 11 Sodium hydroxide11 12 Sodium carbonate and Sodium hydroxide The sample of the enzyme haswith Sephadex G-25.

The results obtained are shown in FIG. 2 in which the activity at eachpH value is shown as relative activity calculated on the assumption thatthe activity at pH 9.0 is 100. As clearly shown in the drawing, theoptimum been previously desalted pH values of the enzymeare found at pH4.5 pH 7 and pH 9, thus the optimum pH range is very broad.

3. Stable pH:

The enzyme solution (0.01 ml) desalted with Sephadex is mixed with 0.1ml of various kinds of buffer solutions containing l,u mole of CaCl as astabilizer.

The resulting mixture is heated at 60C for 30 minutes and then 0.2 ml ofa buffer solution of pH 9.0 and 0.2 ml of a substrate are added to themixture, thus the residual activity has been determined. The results areshown in the following Table 8.

Table 8 pH Buffer Solution Residual Activity(%) 4 Acetic acid 0 6 Trismaleate 100 I0 Na CO NaHCO Table 9 Temperature Time Residual Activityminutes 50 l 5 100 50 30 100 55 l 5 60 l 5 l0 5. Inhibition byTemperature:

The same enzyme solution of pH 8 as shown in (3) is prepared and 5 m Mof Ca is added to the solution.

The solution is heated for 30 minutes varying the temperature as shownin the following table. The residual activities are determined andresults obtained are shown in the following Table.

Table 10 Temperature Residual Activity. pH 4 pH 9 CaCl (M) is added tothe-culture broth of Bacillus sp. No. 38-2 (ATCC 21783) to adjust the pHvalue to and produced precipitates are removed by centrifuge. /2 acetoneis added to the culture broth to form precipitates.

The resulting precipitates are collected and dissolved in water. Afterdialysis overnight, the solution is concentrated withpolyethylene-glycol.

After gel-filtration chromatography with Sephadex G-IOO, activefractions are collected.

The enzyme has been adsorbed on DEAE cellulose column equilibrated with10 m M of Tris-HCl buffer solution containing 1 m M CaCl at pH 8.5 andthen the adsorbed enzyme is eluted varying the concentration of CaClfrom 5 m M to 50 m M. Usually about 40 m M is used.

The active fractions are collected and purified by gelfiltration usingSephadex G-75, to obtain the final product.

Two curves showing the relation between the enzyme activity and the pHvalue which have been measured before and after purification areessentially the same.

7. Homogeneity of the Enzyme: I

The homogeneity of this enzyme has been proved by the followingobservations:

i. Ultra centrifugal analysis gave a single peak of sedimentationconstant at approximately 4.

ii. A single peak of the activity was observed by gelfiltrationchromatography. 1

iii. Disc electrophoretic analysis at pH 8.3 shows rrionodisperse.

iv. The ratio of the enzyme activity at pH values 4 and 9 does notchange, even in the partially denatured enzyme by heating.

The comparison of the physicochemical properties of enzyme disclosedabove and that of the known saccharifying and liquefying amylasesproduced from Bacillus subtilis (Ref: Advances in Applied Microbiology,7, p. 293, 1965) is shown in the following Table ll.

By paper chromatography of the products, a small amount of a series ofoligosaccharides such as maltose, maltotriose, maltotetraose etc. wereobserved. From the facts these enzymes have been determined to beliquefying amylases.

2. Optimum pH:

The optimum pH of the enzymes produced from Bacillus sp. No. 135 and No.169 have been determined by measuring activities at various pH values asdescribed above.

The enzyme used has been previously desalted with Sephadex 6-25. Theother conditions have been disclosed above.

Each pH value used has been achieved by use of the following buffersrespectively.

The results are shown in FIG. 3, in which the activity at each pH valueis shown as relative activity calculated on the assumption that theactivity at pH 10.5 is 100.

As clearly shown in the drawing, both optimum pH values of the enzymesfrom Bacillus sp. No. 135 and No. 169 are found at 10.5.

3. Stable pH:

The pH range in which the activity of .the enzymes can be maintained instable has been studied.

The enzyme (0.01 ml) which has been desalted with Sephadex is mixed with0.1 ml of various kinds of buffer solutions containing 1.0 1. mole ofCaCl2 as a stabilizer.

The resulted mixture is heated at C for 15 minutes and then 0.2 ml of abuffer solution having pH 10.5 and 0.2 ml of a substrate are added, thusthe residual activity has been observed respectively.

The results of the test are shown in the following Table 12.

Table 11 Thermal Stable Optimum Stabilization Hydrolysis Enzyme Typestability pH range pH by Ca of starch Amylase Liquefying 90C 4.8-10.85.4-6.0 35% from Saccharify- B. subtilis ing 5570C 4.0-7.8 4.8-5.2Amylase 4.5 from Liquefying 60-70C 5 10 7.0 15% B.sp.No.38-2 (in thepre- 9.0

- sence of Ca) Note:

* stable unstable ll. Bacillus sp. No. (ATCC 21595) and Bacillus sp.Table 12 No. 169 (ATCC 21594): Residual 1. Substrate Specificity: pHBuffer Activity,

The enzymes produced by cultivation of the above 4 Acetic acid 0identified two microorganisms under the specific conmaleate ditionsdescribed above are active to starch and reduce s 7 51 the starch-iodinereaction. However the increase of re- 10 a ar a 47 11 Na CO .Na0l-l 10ducing activity is little.

Table 13 Amount of Ca Residual Activity,

O I 0.15 1. mole 0 0.25 p. mole 10 0.5 p. mole 25 1.0 u mole 54 L p.mole 40 From the above examination, we found that no increase ofactivation by addition of Ca could be observed, whereas the thermalstability had been improved.

Judging the physicochemical properties of the enzymes from Bacillus sp.No. 135 and 169, these enzymes have characteristic by the fact that theyhave the optimum pH 10.5.

Thus we confirmed that these enzymes are novel amylases.

III. Bacillus sp. No. 13:

1. Substrate Specificity:

The enzyme produced by cultivation of the above identified microorganismunder the specific conditions described above is active to starch andreduces the starch-iodine reaction. However the enzymme does notincrease reducing power at pH 10.5 and it produces cyclodextrin. Thusthe enzyme has been determined to be a cyclodextrin-glycosyl-transferaseand also to be a liquefying amylase. The amylase reduces starch-iodinereaction and increases the reducing power at pH 4.5.

2. Optimum pH:

The optimum pH of the enzyme has been determined by measuring activitiesand yield of cyclodextrin at various pH values by means of the abovedescribed method.

Each pH value has been achieved by use of the following buffersrespectively.

pH Buffer 4 5 Acetate 5 8.5 Tris maleate 9 l l Glycine-NaOH ll 12 NaHCO-NaOH The sample of the anzyme has been previously de-' salted withSephadex G-25.

The results obtained are shown in FIG. 4, in which 60 and from the curveb it is noted that the optimum pH value for production of amylase liesat 4.5.

3. Stable pH:

The pH range in which the activity of the enzyme can be maintained instable has been studied.

The enzyme solution(0.0l ml) desalted with Sephadex is mixed with 0.1 mlof a buffer solution listed in the following table and the mixture isheated at 50C for minutes. Then 0.2 ml of buffer solution of pH 10.0 and0.2 ml of substrate are added to the heated solution. Thus the residualactivities at various pH values are observed. The results obtained areshown in the following Table. l

Table 14 pH Buffer Residual Activity. 7:

4 Acetate 0 5 l0 6 6O 7 Tris maleate 65 8 80 10 N'a CO,.l laHCO 60 l lNa C0 1O 4. Thermal Stability (Conditions for Inactivation):

The th'ermal stability of the enzyme has been studied.

The same enzyme solution of pH 7 as shown in the above paragraph (3) isprepared. The inactivation of the solution has been observed by keepingthe solution at various temperature for 10 minutes respectively.

The results obtained are shown in the following Table 15.

Table I5 Temperature Time Residual Activity C minutes 7:

5. Inhibition, Activation and Stabilization: In same enzyme solution ofpH 7 as disclosed in (3) is prepared.

Various amount of Ca is added to the solution and the increase of theactivity has been studied. Any increase has not been observed.

Then the thermal stability of same solution has been studied by heatingthe solution comtaining 5 mM Ca at different temperature for 20 minutesand measuring the residual activity of the solution. The resultsobtained are shown in the following Table 16.

Table 16 Temperature. C

6. Purification of the Enzyme:

CaCl (5M) is added to a culture broth of the microorganism to formprecipitates and which are removed from the liquid by centrifuge. A halfvolume of acetone is added to the filtrate to form precipitates.

The resulting precipitates are collected and dissolved in water. Afterdialysis overnight the solution is concentrated with polyethyleneglycol.

After gel-filtration chromatography with Sephadex G-100, active factionsare collected. The enzyme is adsorbed on DEAE cellulose columnequilibrated with m M of tris-HCl buffer solution of pH 9.0 containing10 m M CaCl and then the adsorbed enzyme is eluted by varying theconcentration of NaCl from zero to 0.5 M. Usually the enzyme can beeluted at the concentration of about 0.1 M.

Active fractions are collected, the collected solution is gel-filtratedwith Sephadex G-75 to obtain the final product.

Two curves showing the relation between the enzyme activity and the pHvalue which have been measured before and after purification aresubstantially same.

7. Range of Working Temperature:

The activity of the enzyme has been measured at various temperatures andwe found that the optimum working temperature of the enzyme is in therange of from 45C to 50C.

8.'Molecular Weight of the Enzyme:

The molecular weight of the enzyme is determined by means ofGel-filtration method and found being about 60,000.

9. lsoelectric Point:

The isoelectric point of the enzyme has been examined by means of theelectrophoresis using filter paper and found that it is about 4.5.

10. Elementary Analysis:

C: 47.8%, H: 7.2%, S: 0.6%,

N: 15.4%, Ash: 0.9%.

IV. Bacillus sp. No. 17-1 (ATCC 31007):

1. Substrate Specificity:

The enzyme produced by cultivation of the above identified microorganismunder the specific conditions described above is active to starch andreduces the starch-iodine reaction. However no increase of reducingpower can be observed. Almost all of the final products of the enzyme isfound to be cyclodextrin.

2. Optimum pH:

The optimum pH of the enzyme has been determined by measuring activityof the enzyme at various pH values by means of the above describedmethod.

Each pH value listed has been achieved by use of the following buffersrespectively:

Buffer 4, 5 Acetate 6, 7, 8 Phosphate 9, l0 Borate l0, l1 l2GlycineNaOHNaCl The sample of the enzyme has been previously desaltedwith Sephadex G-25.

The results obtained are shown in FIG. 5, in which the curve a showsyield of cyclodextrin at various pH values and the curve I; showsrelative activities of the amylase at various pH values.

From the curve a, it is noted that the optimum pH value for productionof cyclodextrin lies between 10 16 and 10.5 and from the curve b it isnoted that the optimum pH value for production of amylase lies at 4.5.

3. Stable pH:

The pH range in which the activity of the enzyme can be maintained instable has been studied.

The enzyme solution (0.01 ml) desalted with Sephadex is mixed with 0.1ml of a buffer solution listed in the following table and the mixture isheated at 55C for 10 minutes. Then 0.2 ml a buffer solution of pH 10.5and 0.2 ml of substrate are added to the heated solution. Thus theresidual activities at various pH values are observed. The resultsobtained are shown in the following Table 17.

4. Thermal Stability (Conditions for Inactivation):

About 20 30% of the activity of the enzyme has been lost by heating at55C for 10 minutes at pH 10 and almost all of activity has been lost byheating at 60C for 10 minutes.

5. Inhibition, Activation and Stabilization:

We examined the influence of addition of Ca on increase of the enzymeactivity. No increase of the activity can be found, but the thermalstability has been improved by addition of Ca.

Thus an enzyme solution having pH 10 is prepared and the solution isheated at 55C for 20 minutes, and the residual activity has beenexamined by varying the amount of Ca in the solution.

The results are shown in the following Table 18.

Table 18 Amount of added Ca. umole Residual Activity,

6. Purification:

Purification of the enzyme can be carried out by the same methoddescribed in the purification of the enzyme from Bacillus sp. No. 13(ATCC 31006).

7. Range of Working Temperature:

The enzyme activity has been measured at various temperatures and wefound that the optimum working temperature of the enzyme lies in therange of 50C to 55C.

8. Molecular Weight:

The molecular weight by gel-filtration method has been found to be about50,000 to 60,000.

9. Isoelectric Point:

The isoelectric point of the enzyme has been examined by means of theampholine electro-focusing method and we found that it is at pH 4.5.

10. Elementary Analysis:

C: 48.0%, H: 7.3%, S: 0.65%,

N: 15.7%, ash: 1.01%.

Summing physcochemical properties of amylases produced from Bacillus sp.No. 38-2 (ATCC 21783), Bacillus sp. No. 135 (ATCC 21595), Bacillus sp.No. 169 (ATCC 21594), Bacillus sp. No. 13 (ATCC 31006) and Bacillus sp.No. 17-1 (ATCC 31007), these amylases are characteristic in that theyhave the optimum pH value for producing cyclodextrin in the range offrom 9 to 10.5, particularly from to 10.5. Further we found that theseamylase are characteristic in that the ratio of relative activities atpH 4.5 and pH 10 are specific as shown in the Table 19.

Table 19 optimum PH of Ratio of amylase activities From thesecharacteristics, we have determinedthat these amylases are novel ones.

The invention of the present application relates to a process forproducing cyclodextrin characterized by use of a specific alkalineamylase under a specific pl-l condition.

The alkaline amylase to be used must be selected from amylases having anoptimum pH value within the range of from 7 to 10.5, preferably from 8to 10.5, more preferably from 10 to 10.5.

The preferable amylase is a cyclodextrin glycosyltransferase having theoptimum pH value of from 8 to 10.

The alkaline amylase to be used is preferably selected from amylasesproduced by cultivation of a microorganism selected from the groupconsisting of Bacillus sp. No. 38-2 (ATCC 21783), Bacillus sp. No. 135(ATCC 21595), Bacillus sp. No. 169 (ATCC 21594), Bacillus sp. No. 13(ATCC 31006) and Bacillus sp. No. 17-1 (ATCC 31007).

The pH condition to be used according to the present invention shall bewithin the range of pH 6 to 10.5, particularly within the range of pH9.0 to 10.5.

According to the present invention, any kinds of starch may be used, anda preferred starch is potatostarch.

The process of the present invention can be carried out as follows:

Starch is thoroughly liquefied, preferably by NaOH. After adjusting thepH value of the starch solution to 6.0 10.5, preferably to 9.0 10.5, thealkaline amylase disclosed above is added to the solution and thesolution is maintained at the optimum working temperature for sufficienttime to produce cyclodextrin. The optimum working temperature depends onthe kind of the alkaline amylase used as described above. The productionof cyclodextrin is usually accomplished in twelve hours.

We have confirmed that the cyclodextrin obtained according to theprocess of the present invention has the same physicochemical propertiesas that of the known cyclodextrin.

The physical and chemical properties of the cyclodextrin of the presentinvention are as follows:

1. Elementary Analysis:

C: 44.4%, H: 6.1%, O: 49.5%.

2. Molecular Weight:

Crude product 1200 i a-dextrin fraction: 970

B-dextrin fraction: 1.140

'y-dextrin fraction: 1.300

3. Melting Point:

200C (as acetylated) 4. Optical Rotation [a] D a-dextrin fraction:

B-dextrin fraction:

'y-dextrin fraction:

5. Ultraviolet Absorption Spectrum:

No characteristic 6. Infre-red Absorption Spectrum:

Almost same as that of commercially available a-dextrin.

7. Color Reaction:

oz-dextrin fraction: Give blue color by iodine Bq-dextrin fractions:hardly iodine reaction, give yellowish brown or reddish brown color.

8. Crystallography:

a-dextrin fraction: Hexagon or blade.

B-dextrin fraction: Parallelogram.

'y-dextrin fraction: Quadrilateral.

9. Acidity:

Neutral.

10. Color:

White.

11. Reducing Power:

No reducing power:

Glucose, maltose and malt-triose are produced by Taka-a-amylase. But noeffect by an enzyme which decomposes poly or oligosuccharides from thechain termini.

As disclosed above, the cyclodextrin produced according to the processof the present invention contains (1,13 and 'y dextrin fractions.

The enzymes to be used according to the present invention have betterthermal stability higher by 15C to 20C than that of the knowncyclodextrin producing enzyme. Therefore the process of the presentinvention is extremely useful and effective.

The cyclodextrin produced by the process of the present invention hasvarious uses. Particularly it is useful for manufacture of sweeteners,such as millet jelly, because the cyclodextrin glucosyltransferase hastransferase activity.

For instance, a novel and useful sweetener can be produced as follows:

10 gs. dextrin or starch is mixed with 3 gs. sucrose and the mixture isdissolved in water. 100 ml of the enzyme solution containingcyclodextrin-glucosyltransferase is added to the solution and thesolution is allowed to stand at a temperature of 37C overnight.

Unreacted dextrin is removed with trichloroethylene, the solution isconcentrated to obtain 10 gs. of a syrup like millet jelly. We foundthat the product comprises minor sucrose and major compound of sucroseand glucose and has sufficient sweetness for use as a sweetener.

The invention will be explained the following examples, but we do notintend to restrict the invention by them.

EXAMPLE 1 gs. of potato starch is mixed in 100 mls of 1N NaOH solutionand the resulting mixture is stored in a refrigerator over-night tothoroughly liquefy the starch.

100 mg of the alkaline amylase which has been produced by cultivation ofBacillus sp. No. 38-2 (ATCC 21783) is added to the starch solution whichhas been adjusted to pH 9.0. After standing the solution at 34Covernight, any insoluble substance is removed and the product isprecipitated by addition of trichloroethylene.

The collected precipitates are thoroughly washed and trichloroethyleneis removed by heating.

By recrystallization from aqueous propyl alcohol containing 60% ofalcohol, 8 gs. of cyclodextrin is recovered as white crystal.

EXAMPLE 2 The same process explained in Example 1 is repeated exceptthat the alkaline amylase from Bacillus sp. No. 135 (ATCC 21595) is usedinstead of the alkaline amylase from Bacillus sp. No. 38-2.

5 gs. of cyclodextrin has been obtained as white crystal.

EXAMPLE 3 The process explained in the Example 1 is repeated except thatthe alkaline amylase from Bacillus sp. 169 (ATCC 21594) is used as thealkaline amylase.

4.5 gs. white crystalline cyclodextrin has been obtained.

EXAMPLE 4 l0 gs. of potato starch is mixed in 100 ml of 1 N NaOHsolution and the resulted mixture is stored in a refrigelator overnight,to thoroughly liquefy the starch.

mg of the crude enzyme powder which has been produced by cultivation ofBacillus sp. No. 13 (ATCC 31006) is added to the solution which has beenadjusted to pH 10.0. After standing the solution at 34C over night, anyinsoluble substances are removed and the product is precipitated byaddition of trichloroethylene.

The collected precipitates are washed thoroughly and trichloroethyleneis removed by heating.

By recrystallization from aqueous propylalcohol containing 60% ofethanol, 7.5 gs. of white crystalline cyclodextrin has been obtained.

EXAMPLE 5 The process described in Example 4 is repeated except thatsubstituting the a crude enzyme powder produced by cultivation ofBacillus sp. No. 17-1 (ATCC 31007) is used as the alkaline amylase.

7.7 gs. of white crystalline cyclodextrin has been obtained.

We claim:

1. A process for producing cyclodextrin which comprises subjectingstarch to the activty of a cyclodextrin glycosyltransferase produced bycultivation of a microorganism selected from the group consisting ofBacillus sp. No. 38-2 (ATCC 21783), Bacillus sp. No. (ATCC 21595),Bacillus sp. No. 169 (ATCC 21594), Bacillus sp. No. 13 (ATCC 31006) andBacillus sp. No. 17-1 (ATCC 31007), at a pH of 6.0 10.5 and recoveringsaid cyclodextrin.

2. A process according to claim 1, wherein the reaction is carried outat a pH between 8 and 10.5.

3. A process according to claim 2 wherein the reaction is carried out ata pH between 8 and 10.

4. A process according to claim 1, wherein said cyclodextringlycosyltransferase has an optimum pH for producing cyclodextrin ofbetween 8 and 10.

1. A PROCESS FOR PRODUCIING CYCLODEXTRIN WHICH COMPRISES SUBJECTINGSTRACH TO THE ACTIVTY OF A CYCLODEXTRIN GLYCOSYLTRANSFERASE PRODUCED BYCULTIVATION OF A MICROORGANISM SELECTED FROM THE GROUP CONSISTNG OFBACILLUS SP. NO. 83-2 (ATCC 21783), BACILLUS SP. NO. 135 (ATCC 21595),BACILLUS SP. NO. 169 ATCC 21594), BACILLUS SP. NO. 13 (ATCC 31006) ANDBACILLUS SP. NO. 17-1 (ATCC 31007), AT A PH OF 6.0 - 10.5 AND RECOVERINGSAID CYCLODEXTRIN.
 2. A process according to claim 1, wherein thereaction is carried out at a pH between 8 and 10.5.
 3. A processaccording to claim 2, wherein the reaction is carried out at a pHbetween 8 and
 10. 4. A process according to claim 1, wherein saidcyclodextrin glycosyltransferase has an optimum pH for producingcyclodextrin of between 8 and 10.